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Elucidating the Antagonistic Relationship Between Bone Morphogenetic Protein and Activin Signaling Pathways in Osteoprogenitor Cells
Get PDFOsteoporosis is a disease characterized by low bone mineral density due to the rate of bone resorption exceeding that of bone formation. Substantial evidence indicates the Bone Morphogenetic Protein (BMP) pathway promotes bone formation through action of the effectors SMAD1/5/8 while the Activin pathway negatively influences bone mass through action of the effectors SMAD2/3. Recent studies from our lab suggest that BMP and Activin ligands regulate bone mass in a see-saw-like mechanism via competition for a shared pool of receptors, i.e. receptor-level competition. In the present study we seek to test this hypothesis in vitro via signaling responsiveness assays using pathway-specific western blot analyses in the osteogenic cell line W-20-17. We first confirmed that W-20-17 cells respond to exogenous stimulation by BMP2 and Activin-A. Then, we administered recombinant versions of naturally-occurring extracellular ligand traps for BMP2 or Activin ligands (Noggin and Follistatin, respectively) to examine basal antagonism between these pathways. This revealed that, under basal conditions, SMAD1/5/8 activation is repressed by Activin signaling; interestingly, the converse relationship was not observed. To determine the molecular mechanism allowing for this relationship, we treated W-20-17 cells with SB431542, which is an intracellular inhibitor of Activin signaling that functions downstream of receptor engagement, and found no effect on SMAD1/5/8 activation. Collectively, our results suggest Activin-mediated repression of BMP signaling is ligand-dependent but occurs upstream of SMAD2/3 activation. Current studies seek to identify the specific Activin ligand(s) responsible for this effect; gene expression analyses indicates that W-20-17 cells express multiple Activin subunits including Inhβa and Inhβb. Additionally, overpression studies are ongoing to determine if receptor-level competition is involved in mediating these effects. Collectively, our study seeks to elucidate the mechanism(s) that regulate antagonism BMP and Activin signaling pathways to identify novel opportunities for safer and more effective therapies for low bone mass in humans
Welcome / Opening Remarks
No full textIntroduction by Director for Center for Innovation in Technology, Elizabeth Osika
Identification of Commercially Available Antibodies that Block Ligand Binding by BMPR2
Get PDFOsteoporosis, a disease of low bone mineral density, affects 10 million Americans and triggers significant health problems and considerable socioeconomic burdens. Current treatments for osteoporosis have significant limitations, necessitating identifying new treatment strategies via building a better understanding of the endogenous mechanisms regulating bone mass. A recent study demonstrated that removal of the BMP type 2 receptor (BMPR2) in skeletal progenitor cells of Bmpr2-cKO mice during embryonic development leads to reduced age-related bone loss by sustained elevation in bone formation rate. This present study sought to advance the translational potential of the genetic model by identifying antibodies that neutralize the ligand-binding function of the BMPR2 extracellular domain (BMPR2-ECD). This study first established a modified, cell-free immunoprecipitation assay wherein the ligand BMP2 was pulled-down by BMPR2-ECD conjugated to Protein G beads; the unbound BMP2 (found in the supernatant) was subsequently quantified by ELISA. This yielded a standard assay wherein approximately 2 ug BMPR2-ECD leads to a 70% reduction in BMP2 signal. Next, the neutralizing ability of 3F6, a mouse monoclonal antibody raised against the ligand-binding region of BMPR2, was examined and was found to cause a dose-dependent inhibition of BMPR2-ECD ligand-binding. Given the ascites preparation of 3F6, specificity of this assay was confirmed by demonstrating that ligand-binding activity of BMPR2-ECD is unchanged in the presence of non-specific, negative-control ascites. Using these results as a guide, 1F12, another mouse monoclonal antibody raised against the ligand-binding region of BMPR2, was evaluated and was also found to neutralize the ligand-binding function of BMPR2-ECD. In contrast, no effect on ligand-binding function of BMPR2-ECD was observed with 9A10 even though this mouse monoclonal antibody is also raised against the ligand-binding region of BMPR2. These results provide proof-of-concept data for future studies evaluating inhibition of BMPR2 function in vivo as a means to reduce age-related bone loss
The Illusion of Control: Reinvigorating Colonial Desire Through Fantasy Football’s Procedural Rhetoric
Get PDFThis essay examines the online game of fantasy football as a collection of rhetorical procedures that present a particular ideology through how the game is played. Unlike traditional forms of rhetoric, Ian Bogost argues that games are rhetorically unique because they make arguments using procedures, or programmed processes that require the audience to take distinct and discrete actions. I argue that the procedures of fantasy football, from the transformation of human action into numeric representation to trading players with other fantasy owners, are processes that bear the marks of its dominant messages: commodification and ownership. The game of fantasy football is specifically programmed for gamers to think like an NFL owner, where controlling players is programmatically established as the preferred method of operation and the spoils of competition are more likely to come to those gamers who primary view their players as tradeable objects. This relationship between subject and object operates as a kind of colonial logic, rearticulating an already troubling relationship that the NFL holds with America’s plantation past. However, despite its admittance as fantasy in the name itself, the procedures of fantasy football embolden a fetishized, real-life connection between the NFL and fantasy gamers. While fantasy football’s procedures invite owners to exercise control over NFL players, gamers soon realize that they have little impact on the outcome of games. Rather than discouraging the fantasy community from participating in the game, I argue that this illusion of control rearticulates the bond between the NFL and its fans and rejuvenates a colonial desire
Bone Morphogenetic Protein-Based Therapeutic Approaches.
Get PDFBone morphogenetic proteins (BMPs) constitute the largest subdivision of the transforming growth factor (TGF)-β family of ligands and exert most of their effects through the canonical effectors Smad1, 5, and 8. Appropriate regulation of BMP signaling is critical for the development and homeostasis of numerous human organ systems. Aberrations in BMP pathways or their regulation are increasingly associated with diverse human pathologies, and there is an urgent and growing need to develop effective approaches to modulate BMP signaling in the clinic. In this review, we provide a wide perspective on diseases and/or conditions associated with dysregulated BMP signal transduction, outline the current strategies available to modulate BMP pathways, highlight emerging second-generation technologies, and postulate prospective avenues for future investigation
The Transcriptional Co-Repressor TLE3 Regulates Myogenic Differentiation by Repressing the Activity of the MyoD Transcription Factor
Get PDFSatellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity
Uncommon Solutions to Common Problems in Canvas
No full textThis session puts you in the drivers seat. Steve and Tom will be providing solutions to the questions and problems you have experienced with Canvas. In addition, they will demonstrate novel ways to use audio and video, how to use master paths, and explore the learning mastery gradebook
Jigsaw Surveys & Learning Logs: Using Qualtrics and Canvas for Student-Centered Learning
Get PDFThis presentation describes how two educational technology tools were used to enhance student-centered learning. These tools, an online Qualtrics survey completed in tandem with a jigsaw discussion activity and an online reflective learning log on Canvas, significantly contributed to cooperative learning in small group discussions, promoted individual reflection about course themes, and provided personalized information about student progress and challenges that informed instructor feedback and guidance. What has been learned through this process can be used across disciplines to improve student engagement